PSL 7660

November, 2012

NeuroImmunology
Prahlad Parajuli, Ph.D.
Assistant Professor
Department of Neurosurgery
pparajuli@med.wayne.edu

Todays Lecture
An overview of the Immune System
Concept of Immunological Tolerance and Autoimmunity
Immune cells in the CNS
Immunologic aspects of neurologic disorders
Immunotherapy of neurologic disorders
Immune system in CNS and Neural plasticity
Immunology/Immunotherapy of Malignant Gliomas

Immune
System..1
Innate

Acquired

antigen non-specific
specific

antigen

Granulocytes (PMN leucocytes)

Neutrophil
Eosinophil
BasophilMast cell (Ab-triggered
hypersensitivity)

Monocytes/Macrophages/Dendritic cells

NK cells
T cells (TH, CTL, Treg/Tsup)
recognize processed antigen with MHC
B cells (Antibody producing cells)
recognize native antigen

NK Cell

Innate immunity in the periphery and CNS

Microenvironment

(A) In the face of a peripheral infection, innate immune cells prime and instruct T lymphocytes. Tissue DCs internalize microbial
antigens, process them into peptides, and display them on their surfaces with MHC class II molecules; migrate to draining lymph nodes;
and present antigens to naive CD4+ T cells. DCs direct the quality of the subsequent inflammatory response by decoding distinctive
pathogen-associated signals and transmitting this information to T cells in the form of regulatory cytokines such as IL-12 (for Th1), IL-4
(Th2), or IL-6/TGF- (Th17). In addition, lymph node environmental cues can provide information about the site of infection (gut, skin, or
other). Armed with this information, effector T cells migrate to infected tissues. Upon reactivation, Th1, Th2, and Th17 cells express
phenotype-defining cytokines that act on resident and recruited innate cells, which operate collectively with factors such as complement to
clear the infection. (B) Resident microglia and astrocytes exert multiple functions in the CNS, including protective and restorative
responses to CNS infection or injury. Cytokines and chemokines expressed by resident CNS cells also promote the recruitment of
circulating lymphocytes and myeloid cells from the periphery to assist in pathogen clearance. Innate responses in the CNS cannot directly
initiate adaptive immunity. Innate CNS reactions also occur during neuroinflammatory disorders and utilize many of the same components
as do host defense responses.
Ransohoff & Brown, J Clin Invest, 2012

Families of pattern recognition

receptors (PRRs).

Innate immunity is composed of a number of PRRs and these include Toll-like receptors (TLRs), Nod like receptors
(NLRs), Aim2 like receptors (ALRs) and Rig like receptors (RLRs). TLRs expressed on the cell surface sense bacterial
products while endosomal TLRs sense bacterial and viral nucleic acids resulting in the expression of proinflammatory
cytokines and type 1 IFN. Bacterial products are also sensed by the cytoplasmic NLRs, Nod1 and Nod2 leading to
proinflammtory cytokine expression. NALP3, another member of the Nod family and a component of the inflammasome,
detects bacteria in addition to environmental toxins and ATP to activate caspase 1. This results in the cleavage of pro-IL-1
to IL-1, leading to its release. Aim2 also activates caspase 1 following the sensing of cytoplasmic DNA, to permit IL-1
release. IFI16, another member of the ALRs, also senses cytoplasmic DNA, however unlike AIM2, IF116 activates IFN b
expression. RLRs such as MDA5 and RIGI sense cytoplasmic viral RNA to activate type 1 IFN expression via the adapter
M. Carty, A.G. Bowie / Biochemical Pharmacology 81 (2011) 825837
MAVS.

Pro-inflammatory signaling induced by TLRs, IL-1, and TNF-

Signal transduction pathways triggered by the

binding of ligands to their cognate receptors,
which lead to transcriptional activation through
the action of the nuclear factor B (NF-B),
interferon regulatory factor 3 (IRF-3), and
mitogen-activated protein kinases (MAPKs).
The coordinated activation of these nuclear
factors leads to the transcription of proinflammatory cytokines and a large family of
molecules that participate in the control of the
innate immune response. IRF-3 is involved in
the induction of IFN-, and NF-B mediates
the transcription of its own inhibitor, IB.

Bellavance & Rivest, Imm Rev, 2012

Immune System..2
Activation of TH (CD4) cell and CTL
(CD8) by DCs

CD40L
CD40

The DC-T cell interaction involves three signals

Signal 1: Between MHC molecules containing peptide fragments on the DC and the T-cell receptor
(TCR) on the T cell.
Signal 2: Between costimulatory molecules (e.g. CD80/86) on the surface of the DC with ligands (e.g.
CD28) on the T-cell surface.
Signal 3: Secretion of bioactive interleukin 12 (IL-12) by the DC.
Together, these signals promote a T helper 1 (Th1) response within responding T cells.

Human MHC (HLA) Complex

A cluster of genes residing on chromosome 6 of human encode for the HLA,

human version of MHC and many other proteins of immune system.
Antigen presenting cells (APC) process and present antigens (9-13
peptides) on MHC molecules:
CD4+ T (Th) cells recognize antigen on MHC class II molecule
CD8+ T (CTL) recognize antigen on MHC class I molecule

Haplotype inheritance and

co-dominant expression of
MHC antigens

Allele-specific motifs in
peptides
eluted from MHC class II
molecules

Immunological

Tolerance

Immunological Tolerance

Immunological tolerance is a state of unresponsiveness that is

specific for a particular antigen

It is induced by prior exposure to that antigen

Prevent inflammatory response to innocuous air-borne and
food antigens (hypersensitivity/allergy)
Prevent immune attack against self antigens (autoimmunity)
There is potential for such attack because the immune system randomly
generates a vast diversity of antigen specific receptors, some of which will
be self-reactive. Cells bearing these receptors therefore must be
eliminated, either functionally (anergy) or physically.

Development of
Central
Tolerance
Positive and Negative
selection of T cells in
the thymus

Peripheral blood cells from a

healthy individual respond to
the self antigen MBP
Experiment suggests
that some self-reactive
T cells escape
elimination in the
thymus.

PPD, purified protein derivative from mycobacterium tuberculosis;

KLH, keyhole limpet hemocyanin;
MBP, myelin basic protein

Peripheral Tolerance
Sequestration of antigen in some tissues
physical or immunological barrier
Brain absence of lymphatic drainage; BBB
T cell death induced by persistent activation
or neglect
Lymphoid dendritic cells contribute to
peripheral tolerance
Regulatory T (Treg) cells and myeloid derived
suppressor cels (MDSCs): major regulatory
mechanisms in privileged sites (anterior

Regulatory Immune cells

& peripheral tolerance
CD4+CD25+FoxP3+ Treg cells: antigen specific,
cytokine (TGF-, IL-10)-dependent
CD4+CD25+CD45RBlow Treg cells: thymusderived, antigen non-specific, contactdependent
CD8+ T cells (Suppressor T cells)
Myeloid Derived Suppressor Cells (MDSCs):
Immature myeloid cells (neutrophils and monocytic cells)
induced by GMCSF+IL-6

B cell Tolerance
Some self-reactive B cells are deleted in the
bone marrow
In the spleen, self-reactive B cells are purged
by a process that (i) induces anergy;
(ii) prevents migration into B cell follicles;
and
(iii) rapidly leads to cell death
Short-lived, autoreactive B cells may, however, overcome anergy by
cross-reacting to high-avidity foreign antigens

Secondary B cells (derived from memory B

cells produced by T cell-dependent
stimulation) are highly susceptible to
tolerance by epitopes presented multivalently

Autoimmunity

Aetiology of autoimmune
disorders
Self-reactive B and T cells persist
even in normal subsets
Autoimmunity is antigen driven
Controls on the development of
autoimmunity (tolerance) can be
bypassed in a number of ways

Regulatoy Mechanisms
controlling autoimmunity

Genetic Factors
in immune-mediated disorders
Autoimmune diseases can occur
in families
-Thyroid antibodies are associated with
abnormalities of the X-chromosome

Certain HLA haplotypes

predispose to autoimmunity

Immune-mediated Disorders and HLA

relative risk
Disease

HLA Haplotype/
Relative risk
allele
of carriers

Multiple Sclerosis

DR2

4.1

Systemic Lupus ErythematosusDR3

Myasthenia Gravis
Narcolepsy
Sjogrens Syndrome

DR3
DQ6
DR3

5.8
2
>38
9.7

Jeanette et al, in Clinical Neuroimmunology, 2nd Edition, 2006

Cryptic self epitopes

do not induce T cell tolerance

Induction of autoimmune TH
cells and autoantibodies by
cross-reactive antigens

Autoimmunity due to
cytokine disregulation

Introduction of a transgene comprising IFN- on an insulin promoter leads to copius

pancreatic expression and secretion of IFN-. This leads to upregulation of MHC class II and
activation of autoreactive T cells by an as yet unexplained mechanism but probably mediated
through uptake of autoantigen by cytokine-activated DCs. The primed T cells now initiate
autoimmune destruction of the cells. That this is a true auto-aggression is shown by the
prompt destruction of newly grafted normal syngeneic islet cells.

Mechanism of Induction of Autoimmunity

Treg cells and Th subsets in

autoimmunity
Antigen and specific cytokine
signals induce the differentiation
of naive T cells into various
subsets of Th (Th1, Th2 and
Th17). While these subsets
produce specific patterns of
cytokines that induce immunity,
Treg cells, naturally occurring
Foxp3+ Treg cells and induced
Foxp3+ Treg cells secrete antiinflammatory mediators such as
IL-10 and TGF- that maintain
immune tolerance and immune
homeostasis.

Awasthi and Kuchroo, Int Immunol, 2009

Immune response
in the CNS

Brain - an immunopriviledged site

Brain is not connected to the lymphatic system no immunosurveillance
BBB prevents large molecules or molecules with high electrical
charge from entering the bain
. However, there is no absolute size limit for larger proteins, except that they tend to cross-over slower
than smaller proteins.
Antibodies normally do not cross the BBB.
Activated T cells, on the other hand, are known to cross the BBB; but the mechanism is unknown.
Bacteria cannot cross the BBB.
Virus crosses the BBB probably by piggybacking on lymphocytes

BBB can be broken down by:

1.
2.
3.
4.
5.
6.

Hypertension (high blood pressure)

Hyperosmolitity
Microwaves
Radiation
Infection
Trauma, Ischemia, Inflammation, Pressure

Blood-brain Barrier

Immunity is an active component of the CNS

Surveillance is critical in the brain - pathogens are
likely breaching the barrier all the time and require
Microglia mediate surveillance and homeostasis in the CNS
immediate eradication.
Leukocytes patrol the healthy brain and spinal cord

Expression of TLR family members in CNS cells

Major TLR agonists

Microglia express all TLRs identified to date, whereas

astrocytes, oligodendrocytes and neurons express a more limited TLR repertoire in comparison.

Hanke & Kielian, Clinical Science, 2011

Toll-like receptor (TLR) signaling

TLRs are transmembrane proteins with a

large extra-cellular domain containing a
cytoplasmic Toll/IL-1 receptor (TIR)
domain. All TLR family members, except
TLR3, signal through the myeloid
differentiation primary-response gene 88
(MyD88) to recruit downstream interleukin
(IL)-1 receptor-associated kinases
(IRAKs) and tumor necrosis factor (TNF)receptor associated factor 6 (TRAF6).

In TLR2 and TLR4 signaling, MyD88 adaptor-like protein (MAL) is required for recruiting MyD88 to their receptors, whereas in others such as TLR5,
TLR7, TLR9, and TLR11, MAL is not required. TLR1 and TLR2 or TLR2 and TLR6 form heterodimers that signal through MAL/MyD88. TLR3 signals
through the adaptor TIR-domain-containing adaptor protein inducing interferon (IFN)-b-mediated transcription-factor (Trif), which recruits and activates
TNF receptor-associated factor-family member-associated NF-B activator-binding kinase 1 (TBK1). In addition to the MAL/MyD88-dependent pathway,
TLR4 can also signal through a MyD88-independent pathway that activates TBK1 via a Trif-related adaptor molecule (TRAM)-Trif-dependent
mechanism. TLR5, TLR7/8, TLR9, and TLR11 use only MyD88 as its signaling adaptor. These kinases ultimately activate transcription factors such as
nuclear factor-B (NF-B) and IFN regulatory factors (IRFs), which result in production of various cytokines such as TNF, IL, and IFNs.
Wang et al. Journal of Neuroinflammation, 2011

TLRs in health (a) and disease (b) in the CNS

Hanke & Kielian, Clinical Science, 2011

Mast cells in the CNS innate immune responses

Like macrophages, Mast cells are normal residents in the CNS, where they are found in close association
with cerebral blood vessels during development and adulthood. Almost 97% of mast cells lie on the
abluminal (brain) side of the blood vessels. Like lymphoblasts, mast cells can move through normal brain
in the absence of inflammation, with rapid entry mediated via blood-brain barrier (BBB) passage. Whether
these two categories of mast cells (resident vs. immigrant) represent different phenotypes remains to be
established.
Mast cells produce a plethora of mediators, which include biogenic amines (histamine, serotonin),
cytokines
[IL-1 to IL-6, leukemia inhibitory factor, TNF-a, IFN-g, TGF-b, GMCSF], enzymes (acid hydrolyzes,
chymase, phospholipases, rat mast-cell protease I and II, trypase), lipid metabolites (prostaglandins,
leukotrienes, platelet-activating factor), ATP, neuropeptides (vasoactive intestinal peptide), growth factors,
e.g., nerve growth factor (NGF), nitric oxide, and heparin. While mediator release via degranulation is a
very rapid process, longerlasting activation results in the release of de novo formed mediators
Skaper et al, FASEB J, 2012

Pathogenic steps leading to the

initiation of bacterial meningitis

Koedel & Pfister, in Clinical Neuroimmunology, 2nd Edition, 2006

Pathogenesis of bacterial infection into

CNS
Bacterial entry into the central nervous
system elicits the release of many
factors from host cells (macrophages,
microglia, astrocytes, endothelial cells
and infiltrating inflammatory cells),
which exacerbate host cellular
responses, resulting in neuronal injury.
+ denotes upregulation of specific host factors.
BBB, bloodbrain barrier;
EAA, excitatory amino acids;
ICAM, intercellular adhesion molecule; MMP,
matrix metalloproteinases;
NO, nitric oxide;
ROS, reactive oxygen species;
TACE, tumour necrosis factor (TNF)-converting
enzyme.

Possible mechanism of HIV entry

into the brain and neuronal injury

Immunopathologic model of HTLVassociated myelopathy/tropical spastic

paraparesis (HAM/TSP)

Pathogenesis of Autoimmune
Disorder
When autoreactive T cells or antibodies are
found in association with a particular
disease, there are three possible
inferences:
- The autoimmunity is responsible for
producing the lesions of the disease
- There is a disease process which, through
the production of tissue damage, leads to
the development of autoimmune response
- There is a factor which produces both the
lesions and the autoimmunity

Evidences in support of

Immunopathogenesis in CNS
Autoimmunity

The most strong support for immunopathogenesis of CNS

autoimmunity comes from their strong association with MHC
class II haplotypes.

Induced autoimmunity:
- Injection of MBP together with Freunds complete adjuvant
can cause encephalomyelitis

Human antibodies can be directly pathogenic:

- In mothers suffering from myasthenia gravis, antibodies to
acetylcholine receptors cross the placenta into the fetus and may cause
transient muscle weakness in the newborn baby

Immune Complexes appear to be pathogenic in systemic

autoimmunity:
- In patients with SLE, complement-fixing complexes of
antibody with DNA and other nucleosome components such as
histones are deposited in the kidney, skin joints and choroid
plexus, and are presumed to produce type III hypersensitivity
reactions

Autoimmunity and infection

Bystander effects
Epitope spreading
Molecular mimicry
Superantigen

Multilevel cross-talk between immune and neuroendocrine systems

Hypothalamus
CRH

Pituitary

CgA, Chromogranin A; ACh, acetylcholine; AVP, arginine vasopressin; CRH, corticotrophin release hormone; NPY, neuropeptide Y; VIP,
vasoactive intestinal polypeptide; FSH, follicle-stimulating hormone; LH, luteinising hormone; ACTH, adrenocorticotrophic hormone
Comite et al., Autoimmunity Reviews, 2007

Brain circuits involved in the control of the

hypothalamicpituitary-adrenal (HPA) axis during systemic immune stimuli
Circulating immune ligands (e.g. interleukin-1) have the ability to
bind to their receptors located on the surface of endothelial cells
of the brain blood vessels to initiate COX-2 and mPGES
transcription through an NF-kB and MAPK pathway. This leads
to PGE2 synthesis and release by the endothelium and pericytes
of the brain capillaries. The PG may therefore diffuse across the
brain parenchymal elements and bind to specific EP receptors
on the surface of the neurons controlling the autonomic
functions. Whether one or more EP receptors participate in the
neuronal
response to PGE2 is still a matter of great debate, but the
neuroanantomy weighs in factor of the EP4 receptor subtype.
This receptor is found in key regions that control fever, HPA axis
activity, and other autonomic functions. However, PGE2 may
inhibit major inhibitory neurons (e.g. GABA) projecting to the
nuclei that control the autonomic circuits via EP3. This particular
receptor is associated with a Gi-coupled protein inhibiting cAMP
upon its activation, which may take place in GABA neurons
that innervate key neurons of the autonomic system. The
resulting consequence of such integrated circuits is the release
of CRF by neurons of the PVN, activation of the corticotroph
cells in the pituitary, and the secretion of GCs by the adrenal
gland.
ACTH, adrenocorticotrophic hormone; CRF, corticotrophin releasing factor; EP1-4, PGE2 receptor subtypes, GABA, inhibitory caminobutyric acid (major inhibitory neurons of the CNS); IL, interleukin; NTS, nucleus tractus solitarius (A2 C2 neurons); PGE2,
prostaglandin of E2 type; PVN, paraventricular nucleaus of the hypothalamus; VLM, ventrolateral medulla (A1 C1 neurons)

Bellavance & Rivest, Imm Rev, 2012

Importance of Hypothalamus-Pituitary-Adrenal axis

in immune response to CNS infections and survival
Infection/Disease

HPA axis
Mortality
Mortality after
Intervention
corticosterone
replacement

Streptococcal cell wallGlucocorticoid receptor

Induced arthritis
antagonist RU486

100%

MBP-induced EAE

22%

Salmonella

Adrenalectomy

Hypophysectomy

Mouse cytomegalovirus
Shiga toxin

80%
100%

Adrenalectomy

Adrenalectomy

60%

13%

5%
100%

20%

10%

Jeanette et al, in Clinical Neuroimmunology, 2nd Edition, 2006

Defects in Cytokine/HPA
feedback loop in autoimmunity

Immunological aspects of
select Neurologic Disorders

Alzheimers Disease (AD)

Narcolepsy

Narcolepsy is a condition most characterized by

excessive daytime sleepiness (EDS)

Estimated prevalence of approximately 0.05% (20-60

per 100,000) in Western countries.

Strong association with HLA DQB1*0602

Majority of patients with narcolepsy are hypocretin

deficient.
Hypocretin measurment is a newly developed diagnostic tool for
narcolepsy

There is no direct evidence for cellular immunity in

narcolepsy

Several extensive studies have not provided evidence

Epilepsy
Autoantibodies to the B peptide (amino
acids 372-395) of glutamate receptor
subtype 3 are found in serum and
cerebrospinal fluid (CSF) of some patients
with epilepsy as the main disease
Anti-GluR3B Abs were not found in patients
whose seizures accompany antiphospholipid syndrome (APS) or Sneddons
syndrome (SNS), who harbored
characteristic anti-phospholipid antibodies
directed against cardiolipin and beta2glycoprotein I
It is suggested that anti-GluR3B Abs are
produced primarily in the periphery, ad then

Systemic Lupus Erythematosus

(SLE)
SLE is a multiorgan autoimmune disease
with a variety of circulating autoantibodies,
the origin of which remains complex.
Neurological abnormalities are common in
SLE patients
Two potential immunopathologic
mechanisms SLE are:
1) immunecomplex mediated vasculitis; and
2) action of autoantibodies to cell
membrane molecules
Anti-DNA antibodies in SLE patients may
cross-react with CNS lipid antigens
Human CNS myelin consists of 70% lipids
and 30% protein (major protein being MBP

Multiple sclerosis (MS)

Multiple sclerosis (MS) is a nervous system disease
that affects the brain and spinal cord.
It damages the myelin sheath, the material that
surrounds and protects the nerve cells. This damage
slows down or blocks messages between the brain
and body, leading to the symptoms of MS. They can
include:
Visual disturbances
Muscle weakness
Trouble with coordination and balance
Sensations such as numbness or prickling
Thinking and memory problems

Multiple Sclerosis
(MS)
Neuropathological
Features

Immuno-patholology of EAE (MS)

Possible role of the immune system in the

pathogenesis of MS
Summary of the possible events leading to tissue damage in MS and therapeutic intervention strategies

Evidence from knockout mice that TLRs are involved in CNS diseases

M. Carty, A.G. Bowie / Biochemical Pharmacology 81 (2011) 825837

Migration of self-reactive immune cells

into the brain

Immune system responds to danger signal

Chemokines are important messengers of the danger signal

The structure of the brain and routes of leukocyte entry

Beneath the skull lie three membranes that enclose the parenchyma of the brain: the dura mater, the arachnoid membrane,
and the pia mater. The latter two enclose the subarachnoid space. (i) Leukocytes can enter across the choroid plexus,
where CSF is produced by the choroid plexus epithelium in the ventricles. CSF containing leukocytes then enters the
subarachnoid space, circulates around the brain, and (ii) exits via the venous sinus to be resorbed by the blood via the
arachnoid villi. (iii) Blood supply to the brain enters in the subarachnoid space over the pia mater, generating the
perivascular space (or Virchow-Robin space). Main arterial branches divide into capillaries, which terminate deep within the
brain, supplying the parenchyma with blood. Leukocytes can potentially enter from the blood (iii), which requires them to
cross the tightly regulated vascular endothelium (i.e., the BBB: the glia limitans, the subarachnoid space, and the pia
mater). Cells can adhere to the endothelium and arrest at any point during this process.
Wilson et al, J Clin Invest, 2010

Immune surveillance via the choroid plexus

The choroid plexus is composed of highly invaginated loops of capillaries and pia mater that reach into the ventricles of the
brain. Cells from the blood and under the influence of chemokines undergo adhesion, rolling, and diapedesis across the
fenestrated capillary endothelium and pia mater of the choroid plexus. The basement membrane and tight junctions of the
choroid plexus epithelium provide a further barrier, the brain-CSF barrier. These modified epithelial cells (Kolmer cells) have
bulbous microvilli that secrete the CSF. Infiltrating leukocytes migrating through these cells enter the ventricles that contain
CSF and circulate around the CNS. The chemokine CCL20 is expressed on the basolateral side of the choroid plexus
epithelial cells, attracting CCR6-expressing CD4+ T cells.
Wilson et al, J Clin Invest, 2010

Chemokine
s
The major role of chemokines is to
act as a chemoattractant to guide the
migration of cells. Cells that are
attracted by chemokines follow a
signal of increasing chemokine
concentration towards the source of
the chemokine.

Proteins are classified into the chemokine family based on their structural characteristics, not just their ability
to attract cells.
All chemokines are small, with a molecular mass of between 8 and 10 kDa. They are approximately 20-50%
identical to each other; that is, they share gene sequence and amino acid sequence homology.
They all also possess conserved amino acids that are important for creating their 3-dimensional or tertiary
structure, such as (in most cases) four cysteins that interact with each other in pairs to create a Greek key
shape that is a characteristic of chemokines

An overview of Chemokines in MS tissue

Chemokines
Chemokine Receptor(s)
Expression in MS CNS tissue
ativated by the chemokine
(active lesions)
CCL2

CCR2

Astrocytes & Inflammatory cells

CCL3

CCR1, CCR5

Microglia & Macrophages

CCL4

CCR1, CCR3, CCR5

Inflammatory cells &
Activated Neuroglia

CCL5

CCR1, CCR3, CCR5

CXCL10

CXCR3

Perivascular cells & Astrocytes

Astrocytes

Mahad et al, in Clinical Neuroimmunology, 2nd Edition, 2006

An overview of Chemokine Receptor(s) in MS tissue

Chemokine
Chemokines that
Receptors activate the receptors

Expression in MS CNS tissue

(active lesions)

CCR1

CCL3, 5, 7, 14, 15, 16, 17 & 23 Newly infiltrating monocytes

CCR2

CCL2, 7 & 13

Macrophages

CCR3

CCL5, 7, 8, 11, 13, 15, 24 & 26 Perivascular lymphocytes

infiltrating monocytes, activated
microglia &
Macrophages
CCR8
CCL1
Phagocytic Macrophages
CXCR3

CXCL9, 10 & 11

Activated microglia &

Perivascular lymphocytes
Mahad et al, in Clinical Neuroimmunology, 2nd Edition, 2006

Th17 cells in autoimmunity (MS)

Association of IL-17 with human autoimmune disease was first

demonstrated in patients of rheumatoid arthritis.
IL-17 had been shown to induce the production of IL-6, IL-8 and
TNF- from the human synovial cells that induce tissue
inflammation.
Similarly, T cell mediated production of IL-17 had been detected
in the active lesions in the brain of multiple sclerosis patients.
Increased frequencies of Th17 cells and IL-17 have also been
shown in the patients of inflammatory bowel disease and
psoriasis.
Altogether, these observations imply that Th17 cells are
required for the induction of tissue inflammation and pathology
during autoimmune diseases.

Awasthi and Kuchroo, Int Immunol, 2009

Th17 cells in MS (EAE)2

Mice deficient in IL-23p19 are resistant to EAE and IL-17-deficient animals

develop a delayed and attenuated disease.

The administration of an IL-17-blocking antibody to mice immunized with

a myelin antigen prevents chemokine expression in the brain and the
subsequent development of EAE

These findings were paradigm changing, since previously, Th1 cells were
considered to be almost exclusively responsible for driving autoimmune
tissue damage

Both IL-17A and F activated tissue cells to promote inflammation by

producing pro-inflammatory cytokines (IL-6, TNF- and IL-1) and
chemokines (CXCL1, granulocyte chemotactic protein 2, CXCL8,
cytokine-induced neutrophil chemoattractant and MCP-1) help neutrophil
infiltration at the site of the target organ.

IL-17 further potentiates tissue pathology by inducing the production of

nitric oxide and matrix metalloproteinases.

Korn et al, J NeuroImmunol, 2007

Myasthenia gravis
Myasthenia gravis (MG) is a disorder
of neuromuscular transmission
characterized by fluctuating weakness
and fatiguability of skeletal muscles.
The basic pathology is a reduced
number of acetylcholine receptors
(AChR) at the postsynaptic muscle
membrane brought about by an
acquired autoimmune reaction
producing anti-AChR antibodies.
Two major clinical forms of MG are
distinguished, ocular MG and
generalized MG.

The role of complement and antigenic

modulation in MG

(A) IgG4 molecules with different specificity

interchange half molecules (one heavy and one
light chain) resulting in mixed bispecific
antibodies in a process termed Fab arm
exchange.

(B) Effector mechanisms of IgG1 anti-AChR

antibodies cause MG. Bivalent binding of IgG1
leads to complement activation and cross-linking
of AChRs. This results in antigenic modulation and
internalization of AChRs. The main immunogenic
region (MIR) is shown in red.
(C) Fab antibody fragments of anti-AChR
antibodies can bind to the MIR and thereby
prevent binding of full-size anti-AChR antibodies.
Fab antibody fragments do not activate
complement and do not cross-link the AChR.
(D) Monovalent IgG4 molecules do not cross-link
the AChR and can protect against IgG1 anti-AChR
antibodies.
(E) Rapsyn anchors the AChR to the membrane
and the cytoskeleton (not shown). Anchored
AChRs are resistant to modulation by bivalent
antibodies even in the presence of activated
complement.

Losen et al, ANAS, 2008

Inflammation and Depression

Pathways by which micro- or macroorganisms can modulate cognitive function and mood (excluding direct infection of the central
nervous system (CNS)). Acute transient inflammation induced by infection with micro- or macroorganisms can activate serotonergic
neurons in the interfascicular part of the dorsal raphe nucleus (DRI) through activation of spinal afferents. This results in an
antidepressant-like behavioral effect demonstrable in standard laboratory models. By contrast, chronic inflammation, perhaps signaling
through vagal sensory afferents, or following entry of cytokines into the CNS through the circumventricular organs where there is no
bloodbrain barrier (e.g. area postrema and/or nucleus of the solitary tract (NTS)), or through selective transport mechanisms, is
associated with inhibition of DRI neuron activity and with depression. Chronic inflammation induced by infection with micro- or
macroorganisms has multiple consequences. First, it alters the microbiota, both directly by local competition, symbiosis or colonization,
or indirectly by modulating the immune system, and so altering the relationship between the gut immune system and the microbiota,
leading to changes in the balance of strains and species, and to changes in microbiota-induced immunoregulation (in addition there
are other poorly understood gut-brain links that also modulate CNS function; see main text). Finally the Old Friends (a subset of the
microbiota, and other organisms not associated with the gut, with which man has a long association) activate immunoregulatory
pathways (regulatory dendritic cell (DCreg) and regulatory T cell (Treg)) leading to termination of inappropriate chronic inflammation
and thus to diminished depressogenic cytokine signals. This figure illustrates multiple potential sites for therapeutic intervention that
are discussed in the main text. Abbreviations: DRI: interfascicular part of the dorsal raphe nucleus; NTS: nucleus of the solitary tract;
Treg: regulatory T cells; Th2: T helper 2 cells.
Lowry et al, Drug Disc Today, 2012

Immunologic Therapies for

Neurologic Disorders

Current and potential treatment of

autoimmune diseases

Immune-modulating therapies for

narcolepsy
2003 prednisone was unsuccessfully tried
at Stanford
Report on the use of intravenous
immunoglobulins (IVIG) with some clinical
improvement was published shortly after.
Use of IVIG in combination with prednisone
has also been reported
There is a need for a multi-center, placebocontrolled study on the efficacy of IVIG

Alzheimers Disease (AD)

Potential mechanisms underlying effects of

immunotherapeutics in models of AD

Brody and Holtzman, Annu Rev Neurosci. 2008

Potential mechanisms underlying effects of immunotherapeutics

in models of Alzheimer disease

Therapy for Myasthenia Gravis

Acetyl cholinesterase inhibitors: improve
muscle function by slowing the natural enzyme
cholinesterase that degrades acetylcholine in the
motor end plate; the neurotransmitter is therefore
around longer to stimulate its receptor.
Immunosuppressive drugs: prednisone,
cyclosporine, etc. may be used. It is common for
patients to be treated with a combination of these
drugs with a cholinesterase inhibitor.
Plasmapheresis: can be used to remove the
putative antibody from the circulation.
Intravenous immunoglobulins (IVIG): can be

Possible mechanisms of action of

IVIg
Intravenous immunoglobulin (IVIg) has increasingly been used for the treatment of
autoimmune and systemic inflammatory diseases including narcolepsy and
myasthenia gravis
IVIg is prepared from pools of plasma of at least 300010000 and sometimes up to
100000 healthy blood donors

Neutralization of pathogenic autoantibodies and the regulation of

autoreactive B-cell clones by anti-idiotypes and disease associated
antibodies

Induction of anti-inflammatory cytokines and cytokine antagonists,

such as IL-1 receptor antagonist

Interaction of IVIg with membrane molecules of B and T

lymphocytes

Interaction of IVIg with dendritic cells

Attenuation of complement-mediated damage

Fc-mediated blockade of Fc receptors

Treatment for MS

Interferon- and glatiramer (Copaxone) are two drugs

currently used to treat relapsing remitting multiple sclerosis
(MS).

IFN- is an anti-inflammatory cytokine.

Studies have also determined that IFN-improves the

integrity of the blood-brain barrier (BBB), which generally
breaks down in MS patients

Glatiramer acetate (formerly known as copolymer-1) is an

artificial protein that resembles a natural myelin protein. It
may prevent the body's immune system from attacking the
myelin coating that protects nerve fibers.

A study which addresses the safety as well as the

Neuroprotective Effect of
Inflammation
Some autoreactive immune cells may
have neuroprotective function:
Myelin-autoreactive T cells show neuroprotective effects in
vivo in animal models
Activated antigen-specific human T cells and other immune
cells produce neurotrophins such as bioactive brain-derived
neurotrophic factor (BDNF), nerve growth factor (NGF) and
glial cell-derived neurotrophic factor (GDNF) in vitro
BDNF is expressed in different tyes of inflammatory cells in
brain lesions of patients with acute dsseminated
leukoencephalopathy or multiple sclerosis
Hohlfeld et al, Neurol Sci, 2006

Neurotoxicity versus
Neuroprotection

Factors mediating effects of immune

cells on neural stem/progenitor cells
Accumulating evidence indicates that immune cells and
NSPCs share several molecular and cellular
developmental and immune regulatory pathways (for
example, production of chemokines, trophic factors,
cytokines, TLRs and stem cell regulators) that together
render these cells able to interact in response to tissue
damage. This is not entirely unexpected, given that the
function of both the inflammatory reaction and the
mobilization of endogenous NSPCs, occurring in the
CNS in response to danger signals, is to limit the extent
of the tissue damage and, possibly, to promote repair.
Thus, a continuous cross-talk between immune cells
and NSPCs (endogenous or grafted) is probably crucial
for homeostasis in CNS and for survival and preserved
function of its cellular components after damage. These
interactions might constitute a developmental relic, as
innate immune cells home to the CNS before
neurogenesis occurs14 and brain architecture becomes
perturbed postnatally in microglia-depleted mice
BDNF, brain-derived neurotrophic factor; CNTF, ciliary neurotrophic
factor; GDNF, glial cell linederived neurotrophic factor; IGF, insulinlike growth factor; IFN, interferon; IL, interleukin; LIF, leukemia
inhibitory factor; PDGF, platelet-derived growth factor; SDF, stromal
cellderived factor; TGF, transforming growth factor; TNF, tumor
necrosis factor; VEGF, vascular-endothelial growth factor.
Kokaia et al, Nature Neuroscience, 2012

Glioma
Immunology/Immunother
apy

The cancer immunoediting concept

DAMP-HIF, HSP
TLRs on NK cells/M/DCs
T cells (adaptive i.r.)

Only adaptive i.r.

R D Schreiber et al. Science 2011;331:1565-1570

Manipulation of Immune System

for Cancer Therapy

ecific
nes (IL-2), NK/LAK cells

Pioneered by Dr. Steven A. Rosenberg

c
Vaccines Dendritic cell based

e both cellular and humoral immune responses

Pioneered by Dr. Ralph Steinman
Nobel Prize in Medicine 2011

ve:
r: Infusion of ex vivo expanded tumor-reactive T cells
al: Monoclonal antibodies

ed: Active followed by Adoptive

Non-specific Immunotherapy
Coleys toxin
During 1950s, Dr. William B. Coley, a New York physician, became intrigued by the
dramatic disappearance of malignant tumors that he observed in cancer patients who had
contracted acute streptococcal infections.
Out of several cancer patients subsequently treated with streptococcus, some showed
remission. However, the treatment was associated with toxicity and mechanism was not
understood.

BCG Stimulates macrophages and NK cells.

Used as a therapeutic agent in some tumors like bladder cancer
Cytokines IL-2 and IL-12 showed occasional success, but with high toxicities.
IFN is more successful in CML and hairy cell leukemia.

LAK cells Autologous lymphocytes (NK and T cells) activated with cytokines (IL-2
and IL-12) demonstrate antigen non-specific and MHC non-restricted
cytotoxicity against tumor cells in vitro.
The adoptive therapy has not been very successful in patients. Tumor- induced
immune suppression may apply.

Antigen-specific
Immunotherapy
Monoclonal antibodies
(Kohler, Milstein and Jerne, 1975; Nobel prize 1984)

Tumor-specific or tumor-associated
antigens
Proteins specific to cancer cells e.g., idiotypes
Mutated/Over-expressed (shared) proteins - e.g., p53, Her-2/neu, EGFR
Altered proteins e.g., MUC1
Fetal proteins e.g., CEA
Differentiation antigens MAGE, GAGE, testes antigens
Viral Antigens HBV, HCV

Monoclonal Antibodies
Examples:
Rituxan (Retuximab) targets CD20 for B cell non-Hodgkins lymphoma
Herceptin (Trastuzumab) targets HER-2 or erbB2 in breast cancer
In 1980s, severe side reactions to murine antibodies termed Human anti-mouse antibodies
(HAMA) response lead to the development of Chimeric murine/human antibodies
(murine Fab + human Fc region)

Monoclonal Antibodies

Applications & Mechanism of

Action
ADCC: Antibody-dependent cell-mediated

cytotoxicity. Recruits NK cells to cause lysis

Bivalent Abs: Binds to anti-CD3 and a TAA (Her-2)
Growth factor/signaling inhibition
e.g., Avastin (bevacizumab), anti-VEGF mAb
Herceptin (Trastuzumab) anti-HER-2
Magic Bullets: Antibodies conjugated to

toxins (diptheria toxin, tetanus toxoid etc) or

radioactive material
ADEPT-Antibody-directed enzyme/pro-drug
therapy. Enzyme converts an inactive pro-drug
into cytotoxic drug in the tumor vicinity

Antigen-specific
Immunotherapy
Tumor Vaccine
Efforts to elicit an effective cell mediated
immune response against tumors

DC-based Active/Adoptive
Immunotherapy

Peripheral blood
Monocytes

GM-CSF+IL-4

DCs
Load tumor Ag
Into DCs

Digestion
Resection of
primary tumor

Tumor
Single cell suspension

Vaccination

T cells
anti-CD3 + IL-2

Leukapheresis

T cell expansion
and Harvesting

Re-infusion, i.v.

Criteria for an effective

Dendritic Cell vaccine design
Aim is to break immunological tolerance and
elicit an immune response against tumors

Appropriate antigen loading of DC

Proper DC maturation
Licensing from CD4+ helper cells for CD8+ T cell stimulation
Circumvent tumor-induced immunosuppression
Depletion of regulatory T (Treg) cells

Criteria for an effective

Dendritic Cell vaccine design
Aim is to break immunological tolerance and
elicit an immune response against tumors
without inducing autoimmunity

Appropriate choice of tumor antigen

Appropriate antigen loading of DC
Proper DC maturation
Licensing from CD4+ helper cells for CD8+ T cell
stimulation
Circumvent tumor-induced immunosuppression
Depletion of regulatory T (Treg) cells

Glioma-associated antigens
Mutated/overexpressed common cancer antigens: p53, HER-2, EGFRvIII
Melanoma-associated antigens MAGE, GAGE
Cancer testes antigens SART, HOM-TES-85, SSX
Other potential antigens SPARC, IGFBP2, GFAP, GLEA2

Some of these antigens such as HER-2, gp100, and MAGE-1 are shown
to be expressed in human glioblastoma and recognized by cytotoxic T
cells.

Defined Tumor-associated
Antigens
Advantages:
Proteins/peptides can be prepared in bulk
Peptide sequence can be altered to enhance the
immunogenecity
by increasing their affinity for MHC/TCR
molecules
Provides a method to monitor antigen-specific
immune response
by standard in vitro cytotoxicity and ELISpot
analyses
Disadvantages:

Whole Tumor Cell Antigens

ntages:
ractionated tumor material will provide all MHC class I a
ss II epitopes without the need for identification of TAA

ultaneous presentation of antigens through MHC class

ss II pathways lead to a diversified immune response

vantages:
cine needs to be custom-prepared
ficult to monitor antigen-specific responses

approach is particularly appealing for gliomas

ery few TAAs have been identified in these tumors
oming popular due to encouraging clinical outcome

Current Clinical Studies

on
the Immunotherapy of
Gliomas

Adoptive Cellular Therapies for Glioma Patients

Center/
Investigator

Therapy/Protocol

Phase
ND, P, R*
Enrollment

WHO Grade*** Clinicaltrials.go References

v identifier

I - 10

R, P

III or IV

NCT01082926

Autologous CMV
Baylor College
specific CTL
of Medicine,
genetically
I/II - 18
Houston, TX
modified to target
/N Ahmed
Her2

ND

IV

NCT01109095

Penn State
University,
Hershey, PA
/K Lucas

Allogeneic, CMV
specific CTL

I/II - 10

IV

NCT00990496

Bao et al

UCLA, Los
Angeles, CA
/L Liau

Alloreactive CTL
and IL-2

1 - 15

III

NCT01144247

Kruse &
Rubinstein

Hoag Cancer
Center,
Newport
Beach, CA
/R Dillman

Autologous
LAK Cells

II - 80

ND

IV

NCT00814593

Dillman et al

Allogeneic T Cells
City of Hope,
modified with
Duarte, CA
chimeric
/B Badie
IL-132 - TCR

* ND, Newly Diagnosed; P, Persistent; R, Recurrent

** World Health Organization (WHO) Grade III: AA, AODG; Grade IV: GBM
Hickeyet al.Journal of Translational Medicine20108:100 doi:10.1186/1479-5876-8-100

Kahlon et al

Ahmed et al

Non-Cell-based Vaccine Trials for Glioma Patients

Duke Univ, Durham,
/D Mitchell

CDX-110 (EGFRviii)
Pept ide Conjugate
+ TMZ Daclizumab

I/II - 20

ND

IV

NCT00626015

Heimberger
et al

Pittsburgh Cancer
Center, Pittsburgh,
/F Lieberman

GAA peptides
+ PolyICLC

0-9

II

NCT00874861

Butowski et
al ****

Pittsburgh Cancer
Center, Pittsburgh,
/F Lieberman

GAA/TT-peptides +
PolyICLC
+ Montanide ISA-51

0-6

II

NCT00795457

UCSF, San
Francisco, /A Parsa

Autologous HSPPC-96

I/II - 50

IV

NCT00293423

UCSF, San
Francisco, /A Parsa

Autologous HSPPC-96
TMZ

II - 63

ND

IV

NCT00905060

Yang &
Parsa

* ND, Newly Diagnosed; P, Persistent; R, Recurrent

** WHO Grade II: A, ODG, MG; Grade III: AA, AODG, MAG; Grade IV: GBM.
*** GAA peptides include: HER-2, TRP-2, gp100, MAGE-1, IL13R alpha, and AIM-2; patients with Brain Stem
Glioma are eligible for enrollment
****GAA peptides include: IL-13Ralpha2, Survivin, EphA2 and WT1-derived peptides; GAA/TT includes helper
peptide derived from tetanus toxoid
Hickey et al. Journal of Translational Medicine 2010 8:100 doi:10.1186/1479-5876-8-100

DC-based strategies for activation

of T cells
Apoptotic
Total
Freeze-thaw Tumor-eluted
Bodies
RNA
Lysate
Peptides
DC-Tumor Fusion
CD40L /IFN-KLH/BCG/CpG
(Maturation)

Vaccination
Immunological Monitoring:

PBMC harvest and ex vivo activation

Cytotoxicity
Proliferation
Intracellular/ELISpot)

Phenotype & IFN-

Cell-based Vaccine Trials for Glioma Patients

Center
/Investigator

Therapy
/Protocol

Phase
Enrollment

ND, P, R*

WHO
Grade **

Clinicaltrials.gov
identifier

Ref.

UCLA, Los Angeles,

Autologous DC
CA
+ Tumor Lysate
/L Liau

I - 36

ND

III or IV

NCT00068510

Liau et al

Cedars-Sinai,
Los Angeles, CA
/S Phuphanich

I - 39

R, P

IV

NCT00576641

***

Duke Univ, Durham,

Autologous DC + Brain Tumor
NC
I - 50
Stem Cell-mRNA
/D Mitchell

IV

NCT00890032

Mass General,
Boston, MA
Autologous Tumor Cells +
/W Curry
Irradiated GM-CSF Producing I - 25
Dana Farber, Boston,
K562 Cells
MA
/P Wen

III or IV

NCT00694330

Autologous DC
+ Synthetic Glioma Peptide

The clinical outcome of these studies were very limited.

Induction of systemic T cell response did not always translate into clinical response.
Clinical response did not necessarily correlate with systemic T cell response

Challenges to a successful
immune response against
brain tumors:
Brain, an immuno-priviledged site
(BBB and BCSFB)
Glioma-induced
immunosuppression

Challenges to a successful immune response

against tumors:

Peripheral Tolerance

Tumor-antigens are self antigens.

These are, therefore, often ignored or tolerated by the immune system

by mechanisms that normally protect the body from autoimmune
responses, including:
Peripheral deletion of antigen-reactive T cells.
Active (antigen-specific) immuno-suppression by regulatory T (Treg) cells
and myeloid derived suppressor cells (MDSCs)
T cell Anergy or functional non-responsiveness to the antigen
Improper antigen-presentation to T cells
However, most of the mechanisms of tolerance are reversible
Successful in vitro activation of autologous CTLs by DCs obtained from peripheral blood monocytes of glioma
patients

Challenges to a successful immune response

against tumors:

immune escape

mechanisms
Reduced or no expression of MHC class I in about
50% of tumor specimens studied evade CTL attack
Reduced expression of NK-activating receptors
evade NK cell attack

Induction of death receptor (FAS, TRAIL)-mediated

apoptosis in tumor-infiltrating lmphocytes
Inhibition of cytolytic activity of T/NK cells by
inhibitory factors (TGF-)
Induction of regulatory T cell (Treg) and MDSC
activity

Mechanisms of immune suppression by malignant gliomas

IL-6

TGF-1

Th17

Treg
Glioma

CTL

DC
MDSC

Current/Future Directions
DNA/RNA based vaccines
Improved maturation of DC using TLR ligands, e.g., CpG DNA
Carbohydrate Antigens e.g., gangliosides GD2
More study on other effector cells (NK-T) and regulatory T cells
More study on immunobiology of brain tumors
Intra-cranial versus systemic imunotherapy for brain tumors
Autologous T cells armed with bispecific antibodies for adoptive immunotherapy
mAbs guide activated T cells to the tumor.
Combination/Corrective Immunotherapy

Reversal of tumor-mediated immune suppression e.g., use of TGF- inhibitors

Depletion of Treg cells and MDSC

combination with agents that sensitizes the tumor cells to T/NK cell induced
apoptosis